Mechanism and inhibition of the phenylethylamine oxidase from Arthrobacter globiformis
Juda, Gregory Alexander.
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Copper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, utilizing O2 as the oxidant with concomitant production of H2O2 and NH3. The discovery that the human vascular adhesion protein (HVAP-1) is a copper amine oxidase has sparked considerable interest in the mechanism and inhibition of these enzymes. With the potential for therapeutic applications, substantial efforts have been made to determine the molecular factors which govern inhibitor sensitivity and selectivity for copper amine oxidases. In order to contribute to this growing body of knowledge, a series of inhibitors were screened for their ability to inhibit enzymatic catalysis in the phenylethylamine oxidase from Arthrobacter globiformis (AGAO). This CuAO was chosen as a model enzyme for these studies due to its ease in expression and purification as a C-terminal Strep-tag II fusion protein. This work describes the kinetic and structural characterization of a new class of highly potent, reversible CuAO inhibitors with inhibition constants in the low nanomolar range. In addition, an investigation of the molecular details of copper amine oxidase catalysis is described; in particular, mechanistic studies of the controversial oxidative half reaction. Studies using the monodentate copper ligand cyanide provided the first spectroscopic evidence for cyanohydrin derivitization of the quinone cofactor in AGAO and led to the proposal of a detailed mechanism for inhibition of CuAO catalysis by cyanide. Lastly, the role of copper during the oxidative half-reaction of amine oxidation in AGAO and Pichia pastoris lysyl oxidase (PPLO) was investigated using the exogenously added copper ligand azide. This work describes the first examination of the effects of azide on both the oxidative and reductive half-reactions of multiple CuAOs. Azide is the first example of a CuAO inhibitor that significantly reduces the catalytic rate for one halfreaction thereby abolishing the kinetic independence of the other half-reaction. Taken collectively, these results show that in the case of some CuAOs it is experimentally impossible to discern the effects of azide on copper mediated enzymatic reoxidation from the inhibition induced during the reductive half-reaction.